*2.4.1 LPS constitutes a chemical and physical protective barrier for the cell*

LPS of Gram-negative bacteria, a major component of the outer membrane, constitute a chemical and physical protective barrier for the cell. LPS consists of the hydrophobic lipid A, a short non-repeating core oligosaccharide and a long distal repetitive polysaccharide termed O-antigen or O-side chain [105]. Complete LPS is characterized by long O-antigen which confers the smooth (S) phenotype on *Salmonella*. The O-antigen is a major component associated with serum resistance. Incomplete LPS devoid of O-antigen leads to rough (R) phenotype, which is of low virulence [106]. Naturally occurring infections are caused by S-phenotype *Salmonella*, which are resistant to complement killing [107, 108]. There is a correlation between the amount, structure, and chain length of the O-antigen and virulence [109]. The long O-antigen of LPS confers on the organism the ability to resist complement-mediated serum killing by sterically hindering the insertion of the membrane attack complement complex (C5b-9) into the bacterial outer membrane [107, 108].

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**Table 4.**

*dissemination.*

*Virulence Determinants of Non-typhoidal* Salmonellae *DOI: http://dx.doi.org/10.5772/intechopen.88904*

**Virulence genes Location Functions** *cirA* Chromosome Colicin I receptor *entABCDEF* Chromosome Enterobactin synthase

*fepABCDEG* Chromosome Outer membrane receptor; iron-enterobactin

*Fes* Chromosome Salmochelin secretion/degradation *FhuABCDE* Chromosome Enterobactin/ferric enterobactin esterase *foxA* Chromosome Ferrioxamine B receptor precursor *FruR* SPI-2 DNA-binding transcriptional regulator

*iroBCDE* Chromosome Salmochelin glycosylation, transport and

*rfbBDFGHIJKMNOPUVX* Chromosome Glucose biosynthesis pathway; O-chain

*MsbA* Chromosome Lipid transporter ATP-binding/permease protein *rfaBCDFGHIJKLPQYZ* Chromosome LPS core biosynthesis protein; transcriptional

*wzxCE* Chromosome Colanic acid exporter; putative LPS biosynthesis

*wzzBE* Chromosome LPS chain length regulator and biosynthesis

*FUR* Chromosome Ferric uptake regulator

*rfc* Chromosome O-antigen polymerase *STM0719* Chromosome Unknown function

*yibR* Chromosome Unknown function *ybdAB* Chromosome Enterobactin exporter EntS

transporter binding protein

activator; O-antigen ligase

glycosyltransferase; O-antigen transporter

processing

protein

protein

Surface expression of O-antigen involves multiple steps: O-antigen biosynthesis in the inner membrane (*rfb*), translocation across the inner membrane by Wzx flippase (*wzx*), polymerization (*wzz*, *rfc* and *rfe*) and ligation on to the preformed Core-Lipid A complex by WaaL ligase (*rfaL*). The Core-Lipid A is translocated independently by the ATP-binding cassette (ABC) transporter MsbA [110, 111]. Complete LPS molecules are then transported to the surface across the periplasm and outer membrane by the Lpt (LPS transport) pathway [111]. Defects in any of the above steps would affect the surface display of the O-antigen and its function. The mutants defective in the biosynthesis of LPS core encoded by the *rfa* loci or the O side chain by the *rfb* loci, are significantly attenuated with a LD50 at least 100 times higher than the parental strain in

*Location and function of the major proteins and virulence determinants contributing to Salmonella* 

Typhimurium possesses two functional *wzz* genes responsible for regulating the chain length of the O-antigen [113]. One is *wzzST* encoding a long LPS with 16–35 O-antigen repeat units and the other *fepE* gene coding for a very long LPS estimated to contain more than 100 repeat units [113]. Either gene product is sufficient for complement resistance and virulence in the mouse model of infection, which reflects a degree of functional redundancy of these two *wzz* genes [113]. Double mutation of these two *wzz* genes resulted in relatively short, random-length

chickens subcutaneously infected with Enteritidis [112].


#### **Table 4.**

*Microorganisms*

SPI-1 gene expression through HilD [101].

**2.4 Systemic infection/dissemination**

siderophores (**Table 4** and **Figure 1**).

**2.3 Intramacrophage survival and replication**

SprB encoded within SPI-1 and regulated by HilA under similar environmental conditions; SprB directly activates SPI-4 gene expression and weakly represses

Similar mechanisms occur inside epithelial cells after intestinal invasion and once bacteria have been internalized by macrophages. Briefly, *Salmonella* cells are localized in the SCV once engulfment is completed. Preserving the SCV membrane integrity plays a crucial role in allowing *Salmonella* replication inside these intracellular niches. These procedures are regulated by T3SS-2 transporting action and its translocon machinery, namely SseBCD complex [77]. Hence, the required effectors which are encoded both inside and outside SPI-2 facilitate the success of *Salmonella* intramacrophage survival. The SPI-2 gene expression is triggered in response to a number of environmental signals mimicking the vacuolar environment of SCV, including stationary growth phase, low osmolarity [102], low concentrations of Mg2+, Ca2+ or PO3 [103, 104], and low pH [76]. The expression of SPI-2 genes is coordinately regulated at both transcriptional and post-transcriptional levels. During the transcription of SPI-2 genes, many two-component regulatory systems are involved, including SsrA-SsrB, OmpR-EnvZ and PhoP-PhoQ as well as transcriptional regulators, namely SlyA and the alternative sigma factor of RNA polymerase RpoE. The main regulatory proteins that act post-transcriptionally are the RNA chaperons, including Hfq, CsrA, and SmpB. The *mgtC* gene located in SPI-3 has been shown to contribute to replication in macrophages. All the men-

tioned virulence determinants can be found in **Table 3** and **Figure 1**.

*2.4.1 LPS constitutes a chemical and physical protective barrier for the cell*

Internalization of the infecting *Salmonella* within SCV is followed by systemic spread through other target organs, such as the spleen and liver. As a prerequisite for spread, the bacterial cells must evade the innate immune system. During this process, serum resistance or resistance to complement-mediated serum killing is a major virulence factor for the development of systemic salmonellosis. It involves three major factors, namely LPS, outer membrane proteins PagC and Rck and

LPS of Gram-negative bacteria, a major component of the outer membrane, constitute a chemical and physical protective barrier for the cell. LPS consists of the hydrophobic lipid A, a short non-repeating core oligosaccharide and a long distal repetitive polysaccharide termed O-antigen or O-side chain [105]. Complete LPS is characterized by long O-antigen which confers the smooth (S) phenotype on *Salmonella*. The O-antigen is a major component associated with serum resistance. Incomplete LPS devoid of O-antigen leads to rough (R) phenotype, which is of low virulence [106]. Naturally occurring infections are caused by S-phenotype *Salmonella*, which are resistant to complement killing [107, 108]. There is a correlation between the amount, structure, and chain length of the O-antigen and virulence [109]. The long O-antigen of LPS confers on the organism the ability to resist complement-mediated serum killing by sterically hindering the insertion of the membrane attack complement complex (C5b-9) into the bacterial outer

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membrane [107, 108].

*Location and function of the major proteins and virulence determinants contributing to Salmonella dissemination.*

Surface expression of O-antigen involves multiple steps: O-antigen biosynthesis in the inner membrane (*rfb*), translocation across the inner membrane by Wzx flippase (*wzx*), polymerization (*wzz*, *rfc* and *rfe*) and ligation on to the preformed Core-Lipid A complex by WaaL ligase (*rfaL*). The Core-Lipid A is translocated independently by the ATP-binding cassette (ABC) transporter MsbA [110, 111]. Complete LPS molecules are then transported to the surface across the periplasm and outer membrane by the Lpt (LPS transport) pathway [111]. Defects in any of the above steps would affect the surface display of the O-antigen and its function. The mutants defective in the biosynthesis of LPS core encoded by the *rfa* loci or the O side chain by the *rfb* loci, are significantly attenuated with a LD50 at least 100 times higher than the parental strain in chickens subcutaneously infected with Enteritidis [112].

Typhimurium possesses two functional *wzz* genes responsible for regulating the chain length of the O-antigen [113]. One is *wzzST* encoding a long LPS with 16–35 O-antigen repeat units and the other *fepE* gene coding for a very long LPS estimated to contain more than 100 repeat units [113]. Either gene product is sufficient for complement resistance and virulence in the mouse model of infection, which reflects a degree of functional redundancy of these two *wzz* genes [113]. Double mutation of these two *wzz* genes resulted in relatively short, random-length O-antigen and the mutant displayed enhanced susceptibility to complement-mediated killing and was highly attenuated in mice [113]. The transcription of *wzzST* gene is independently activated by two-component systems of Typhimurium, PmrA/PmrB (PmrA, sensor; PmrB, response regulator) and RcsC/YojN/RcsB (RcsC, sensor; YojN, intermediate phosphotransfer protein; RcsB, response regulator) [114]. PmrA/PmrB is activated through two pathways: one is directly activated through its cognate sensor PmrB in response to Fe3+ and the other is dependent on the PhoP/PhoQ two-component system in response to low Mg2+. The RcsC/YojN/ RcsB is activated in the presence of low Mg2+ plus Fe3+ [114]. In addition, mutants in a number of genes (*rfaG*, *rfaI*, *rfaL*, *rfaQ*, *rfaP*, *rfbC*, *rfbD*, *rfbJ*, *rfbM*, *rfbP*, *yibR*) necessary for LPS biosynthesis/assembly had severely impaired movement on swimming motility agar [115].
