*2.2.1 SPI-1 mediates contact-dependent invasion of the intestinal epithelium and enteropathogenesis*

SPI-1 codes for several effector proteins that trigger invasion of epithelial cells by mediating actin cytoskeletal rearrangements and hence internalization of the


**175**

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

**Virulence genes Location\* Functions**

*spaOPQRS* SPI-1 Secretion

*SopABDD2EE2 sopB*

(*sigD*)

**Table 2.**

*sodABD* Resistance to oxidative stress

*SsJ* Resistance to oxidative stress

*ymdA* Chromosome Stress response

*\*SPI1–5 are genomic islands on Salmonella chromosome.*

SPI-5 Chloride secretion; promote actin cytoskeletal

macrophages; sif formation

rearrangements, invasion and inhibition of apoptosis of epithelial cells, induction of proinflammatory response and fluid secretion, SCV size, instability, maturation and positioning, nitrate respiration, outgrowth in the intestine; inhibition of vesicular trafficking; replication inside

cells; SCV maturation and positioning; induction of apoptosis;

bacteria. These effectors are translocated into host cell by means of a Type III Secretory System or T3SS-1 [64], which is made up of proteins encoded by the SPI-1, such as *inv*, *spa*, *prg* and *org* [65]. Naturally occurring mutants of *Salmonella* have been found in the environment with a deletion of a vast DNA segment of SPI-1 locus and are deficient for *inv*, *spa*, and *hil* hindering their ability to enter cultured epithelial cells [66]. Mutations leading to a defective secretory function of T3SS-1 led to a 50-fold increase in LD50 following oral administration of Typhimurium in the mouse model [67]. The *prg/org* and *inv/spa* operons encode the needle complex, whereas the *sic/sip* operons encode the effector proteins and the translocon (SipBCD), a pore-forming structure that embeds in the host cell membrane and delivers these effectors to the host cytosol. In addition, several chaperones are also encoded within SPI-1. For example, SlrP mediate ubiquitination of ubiquitin and thioredoxin [68] and one of the SPI-1 regulons, STM4315 (*rtsA*) interferes with the interactions of *S. typhimurium* and host cells [69]. In general, the expression of SPI-1 genes is subject to control by complex regulatory mechanisms involving local regulators such as HilA, iagB and InvF which are necessary for host invasion by *Salmonella* and induction of gastroenteritis [70, 71]. For example, *prgHIJK*, *invA*, *invJ*, and *orgA* are primarily regulated by HilA [71]. In addition, two major global regulatory networks, SirA/BarA and PhoP/PhoQ, indirectly regulate the expression

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

*SprB* SPI-1 Regulation of transcription, DNA-templated

*YejABEF* Chromosome Resistance to AMP, macrophage cytotoxicity

*spvABCD* Plasmid Modifies actin and destabilizes the cytoskeleton of infected

*STM2231* SPI-2 SPI-2 regulator (transcriptional and post-transcriptional)

Host cell signaling

of the invasion-associated genes via HilA [72, 73].

*2.2.2 SPI-2 is essential for survival and replication in macrophage*

The SPI-2 is composed of two segments. The smaller portion contains the *ttrRSBCA* operon, which is involved in tetrathionate reduction, and seven open reading frames (ORFs) of unknown function. The expression of these genes may contribute a growth advantage over the microbiota [74]. The larger portion of this island was shown to be critical for the ability of *Salmonella* to survive and replicate *Virulence Determinants of Non-typhoidal* Salmonellae *DOI: http://dx.doi.org/10.5772/intechopen.88904*


### **Table 2.**

*Microorganisms*

*pipABB2CD pipC*

(*sigE*)

**Virulence genes Location\* Functions**

*iagB* SPI-1 Invasion

*prgHIJK* SPI-1 Secretion

*msgA* Chromosome Unknown function *ompR/envZ* SPI-2 Regulates *ssrAB* expression *orgABC* SPI-1 Pathogenesis; secretion

*Crp* Chromosome cAMP-regulatory protein

*HtrA* Resistance to periplasmic stress *IacP* SPI-1 Posttranslational modification

*hilACD* SPI-1 Promote phop-repressed prgHIJK, sipA, sipC, invF, and orgA;

*Hnr* SPI-2 SPI-2 regulator (transcriptional and post-transcriptional)

*invABCEFGIJ* SPI-1 Secretion and chaperone; promote sipBCDA, sigD and sicA

*phoR/Q* SPI-2 Regulates *ssrAB* expression; down-regulates the transcription

and positioning

*rpoES rpoS* (*katF*) SPI-2 SPI-2 regulator (transcriptional and post-transcriptional);

*sifA* SPI-2 Sif formation in epithelial cells and maintenance of SCV

*sipA* (*sspA*) SPI-1 Stabilization and localization of actin filaments during

*sipBCD* (*sspBCD*) SPI-1 Adhesion to epithelial cells, early macrophage pyroptosis,

*SpaSRQPO* SPI-1 EscU/YscU/HrcU family type III secretion system export

inflammatory response

*sptP* SPI-1 Disruption of the actin cytoskeleton rearrangements

*sspH1H2* Phage Localize to the mammalian nucleus and inhibits NF-κB-

*sirA* SPI-1 SirA/BarA encoded outside SPI-1 activates HilA

*slyA* SPI-2 Regulates resistance to oxidative stress

*slrP* Chromosome Adhesion to epithelial cells

stationary growth phase

membrane integrity *siiCDEF* SPI-4 Translocation; adhesion to apical side of polarized epithelial

*pagACDP* SPI-11 Resistance to AMP, macrophage cytotoxicity

*Prc* Resistance to periplasmic stress

*sicAP* SPI-1 Chaperone for sipBC

*rtsA* Chromosome Activates the expression of the *hilA* gene *sapABCDF* Resistance to AMP, macrophage cytotoxicity

activates the expression of the *hilA* gene

of its master regulator HilA, control *mgtC*

SPI-5 Pathogenesis, effector protein; sif extension; SCV maturation

controls the transcription of the regulatory gene *spvR*; expression of rpoS is induced after entry of *Salmonella* into macrophages or epithelial cells, or in vitro during the

cells; involved in T3SS-1 dependent invasion

inflammatory response and fluid secretion

macrophage autophagy; Adhesion to epithelial cells

invasion, stabilization of VAP, correct localization of SifA and PipB2, SCV perinuclear migration and morphology, promote

apparatus switch protein; antigen presentation protein SpaO

dependent gene expression; SCV maturation and positioning

by antagonizing SopE, SopE2, and SigD, downregulate

**174**

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

bacteria. These effectors are translocated into host cell by means of a Type III Secretory System or T3SS-1 [64], which is made up of proteins encoded by the SPI-1, such as *inv*, *spa*, *prg* and *org* [65]. Naturally occurring mutants of *Salmonella* have been found in the environment with a deletion of a vast DNA segment of SPI-1 locus and are deficient for *inv*, *spa*, and *hil* hindering their ability to enter cultured epithelial cells [66]. Mutations leading to a defective secretory function of T3SS-1 led to a 50-fold increase in LD50 following oral administration of Typhimurium in the mouse model [67]. The *prg/org* and *inv/spa* operons encode the needle complex, whereas the *sic/sip* operons encode the effector proteins and the translocon (SipBCD), a pore-forming structure that embeds in the host cell membrane and delivers these effectors to the host cytosol. In addition, several chaperones are also encoded within SPI-1. For example, SlrP mediate ubiquitination of ubiquitin and thioredoxin [68] and one of the SPI-1 regulons, STM4315 (*rtsA*) interferes with the interactions of *S. typhimurium* and host cells [69]. In general, the expression of SPI-1 genes is subject to control by complex regulatory mechanisms involving local regulators such as HilA, iagB and InvF which are necessary for host invasion by *Salmonella* and induction of gastroenteritis [70, 71]. For example, *prgHIJK*, *invA*, *invJ*, and *orgA* are primarily regulated by HilA [71]. In addition, two major global regulatory networks, SirA/BarA and PhoP/PhoQ, indirectly regulate the expression of the invasion-associated genes via HilA [72, 73].

#### *2.2.2 SPI-2 is essential for survival and replication in macrophage*

The SPI-2 is composed of two segments. The smaller portion contains the *ttrRSBCA* operon, which is involved in tetrathionate reduction, and seven open reading frames (ORFs) of unknown function. The expression of these genes may contribute a growth advantage over the microbiota [74]. The larger portion of this island was shown to be critical for the ability of *Salmonella* to survive and replicate


#### **Table 3.**

*Location and function of the major proteins and virulence determinants contributing to Salmonella macrophage survival and replication.*

inside host cells—both epithelia cells and macrophages—within the SCV [75]. Non-functional SPI-2 mutants are unable to colonize internal target organs such as spleen and liver of mice, although they penetrate the intestinal barrier as efficiently as the wild type strain [76]. These mutants were attenuated by at least five orders of magnitude compared with the wild type strain after either oral or intraperitoneal inoculation of mice [75]. The SPI-2 related events are triggered by the action of effector proteins with its own T3SS known as T3SS-2, which also encodes its proper translocon machinery named SseBCD [77]. Gene sequence similarity to the known components of other T3SS has been used to propose functions for *SsaN*, *SsaR*, *SsaS*, *SsaT*, *SsaU* and *SsaV* as coding for putative proto-channel components, SsaD/SpiB, SsaJ, SsaK and SsaQ appear to code for basal components, whereas SsaC/SpiA may code for an outer ring protein [78]. Generally, SPI-2 contains four types of virulence genes: *ssa* encodes T3SS-2 apparatus; *ssr* encodes regulators; *ssc* encodes the chaperones and *sse* encodes the effectors (**Table 2**) [79, 80].
