**5.2 The SPI1 regulon**

The expression of SPI1 is driven by central transcription factor, SPI1 encoded HilA which is a member of OmpR/ToxR family of transcriptional regulators. The expression of SPI1 regulon is directed by specific blend of environmental signals such as osmolarity, antimicrobial peptides, oxygen, pH and other unidentified signals. These signals are perceived by a set of 2 component regulatory systems: BarA/SirA [17], OmpR/EnvZ [18], PhoBR [19] and PhoPQ [20]. The phosphorylated connected response regulators can promote the expression of either HilD or HilE that in turn either stimulates or represses SPI1-expression.


#### **Figure 2.**

*The SPI1 regulon. The main regulatory factor of SPI1 expression is HilA whose activity is mainly controlled on the transcriptional level and relies on a complex network of transcription factors and two-component regulatory systems. Arrows indicate activation of gene expression. Positive feedback loop are highlighted by bold arrows. Repression is noted as lines with blunt ends. Solid lines represent direct transcriptional regulation. (from Gerlach RG, Hensel M.* Salmonella *pathogenicity islands in host specificity, host pathogen-interactions and antibiotics resistance of* Salmonella enterica*. Berliner und Munchener tierarztliche Wochenschrift. 2007 Jul 1;120(7/8):317).*

### **5.3 SPI2**

*Salmonella* has a second T3SS that is essential for survival within the macrophage and for establishment of systemic infection. Proteins delivered by both type III secretion systems are vital for intracellular survival. The second T3SS is encoded on *Salmonella* pathogenicity island 2 (SPI-2). The activity of SPI2 is needed to establish and maintain the *Salmonella-* containing vacuole (SCV) as an intracellular niche in which *Salmonella* can remain live and replicate.

SPI2 is of nearly 40 Kb in size and comprises of 2 distinct regions [24]:


This second T3SS expressed by intracellular bacteria translocates proteins across the SCV membrane into the macrophage cytosol. With the help of these SPI2 translocated proteins, *Salmonella* escapes intracellular killing by altering the phagosome membrane to tubulate [25]. Phagosome tubulation is dynamic and rapid process and occurs to be dependent on the recruitment of microtubule motors, membrane lipid alteration and the activation of small GTPases, and membrane lipid alteration [25]. Phagosome tubulation is also correlated with the virulence by unknown mechanisms.

A total of seventeen effectors are recognized to be translocated over the SCV membrane into the host-cell cytoplasm, most of them being encoded outside the SPI2-locus [26]. Only 3 effectors are known to be encoded within SPI2 which includes SpiC, SseF and SseG. The SPI-2 translocated proteins, including SifA, SifB, SseJ, SopD2, PipB, and PipB2, localize to the surface of the SCV and either contributes to tubulation or other alterations of the phagosome [27]. The summary of important effectors has been given in **Table 3**.

### **5.4 The SPI2 regulon**

The expression of SPI2 genes is controlled governed by global regulatory system: SsrAB system. It is a typical two-component system that is necessary for SPI2 regulon expression in intracellular bacteria. The main global regulatory systems that affect the expression levels of SPI2 genes are the EnvZ/OmpR and PhoPQ two-component systems, SlyA and Fis [23].


#### **Table 3.**

*Functions of major effectors of SPI-2.*

## **5.5 SPI3**

The SPI3 locus is of size 17 kilobases and is inserted at the selC tRNA gene locus. The primary known virulence determinant is Mgt CB (Magnesium transport system) operon: MisL and Mar T. This determinant is necessary for survival of *Salmonella* in the intra-phagosomal habitat in nutritionally deprived conditions. Mis L, a antitransport protein of SPI3, is identical to the autotransported AIDA-1 adhesin of enteropathogenic *E. coli* (EPEC) while Mar T shows resemblance with Tax R (Toxin gene regulator) of *Vibrio cholerae* and it is implicated in the activation of Mis L [28].

MisL is proved to work as an adhesion [29] and it is vital for the long term persistence of *Salmonella* in the intestine as observed in animal studies. Another autotransporter, ShdA is seen to have a function in adhesion and virulence in case of *S.* Typhimurium.

A high degree of sequential variation exists in SPI3 among different serovars; however it is conserved in cases of *S.* Typhi and *S*. Typhimurium.

### **5.6 SPI4**

The size of SPI4 is identified as 27 Kb. Sequencing of the *Salmonella* Typhimurium genome anticipated that the pathogenicity island constitute of not more than six genes. Hence the genes of the locus SPI4 are named as siiA-F. SiiC, SiiD and SiiF encodes components of type I secretion system which secretes SiiE. This, SiiE is huge protein (approximately 600 kDa) that is known to colonize the bovine intestine [30]. The molecular functions of SPI4 encoded proteins are not known. The role of SPI4 in *Salmonella* virulence was investigated in one of the studies using refined cell culture and infection models, there it was observed that SPI4 contributes to gastrointestinal inflammation in murine colitis model and is also required for adhesion to epithelial cells [31]. De Keersmaecker et al. suggested a role for SPI4 in intra-macrophage survival as shown for SPI2 [32].

SPI4 seems to be highly conserved among different *Salmonella* serovars [33].

#### **5.7 SPI5**

The size of SPI5 locus is nearly 7.6 Kb. It encodes the effector proteins for both the T3SS that is encoded by SPI-1 and SPI-2. Pip A and Pip B are also known to be encoded by SPI5 locus. Pip A is implicated in the development of systemic infection and Pip B is involved in the accumulation of lipid rafts and is a translocated effector of SPI-2 encoded T3SS which is under the control of Ssr AB two-component systems. However, PipB is neither needed for bacterium's intracellular survival nor for systemic virulence as studied in mice [34, 35].

In enteropathogenicity in a cattle infection model, significant attenuation of SPI5-deficient *Salmonella* was observed. However SPI5 mutants showed only a minor virulence defect in mouse model [36].

#### **5.8 SPI6**

The SPI6 locus is also known as '*Salmonella* centisome 7 genomic island' or SCI [37]. It is of size 59 Kb and it has been recognized in *S*. Typhi and *S*. Typhimurium. It is investigated to contain [35]:

1. saf gene which codes for fimbriae

2.pag N gene which encodes for invasion protein

A microarray analysis indicated the conservation of SPI6 among serovars of S. enterica subspecies I serovars was indicated by microarray analysis.

Deletion of SPI6 had no influence on the systemic pathogenesis but decreased invasiveness of the bactetia in tissue cultured cells. SPI-6 was detected to be conserved among serovars of S. *enterica as* indicated by microarray analysis. Some of the portion of SPI-6 that was also identified in subspecies III b, IV, and VII. Further, SPI-6 has shown sequential homology with the genome of *P. aeruginosa* and *Y. pestis* [38].

#### **5.9 SPI7 and SPI8**

The size of SPI7 and SPI8 is approximately 133 Kb and 6.8 Kb respectively. SPI7, also termed as major Pathogenicity Island is specific to *S.* Typhi, *S.* Dublin and *S.* Paratyphi. It encodes for Vi antigen and constitute pil gene cluster that encodes for putative virulence factors. Its genetic organization is very complex and composed of several horizontally acquired elements. It also constitutes few genes of conjugative plasmid-like *tra* and *sam.*. The locus is said to not stable and loss of the capsule can be seen in *S.* Typhi isolates. Additionally SPI7 also encodes a type IV fimbrial adhesin. There exists a sequential homology with few other bacteria like *Xanthomonas axonopodis* and *Pseudomonas aeruginosa* in the case of SPI7 [39].

SPI8 has been identified in *Salmonella* Typhi and the genes located here encode for putative virulence factors, whose exact function has not been reported so far.

#### **5.10 SP19**

The size of SPI-9 locus is nearly 16,281 basepairs. SPI9 from S. Typhi harbors three ORFs (STY2876,STY2877,STY2878) presenting 98% identity with a type 1 secretory apparatus (T1SS) and a single ORF (STY2875) that is similar to a large RTX-like protein exhibiting repeated Ig domains. It encodes for virulence factors of type I secretion system. Furthermore, as it is functional in *S*. Typhi and encodes for adhesion which is induced under conditions of high osmolarity in culture. However it does not participate in biofilm formation [40].

#### **5.11 SP110**

SPI10 has a size of 32.8 Kb and is defined as an insertion at the tRNA leuX gene. It appears to be hyper variable and is a point of insertion for several different DNA fragments. Sef and pef gene clusters which encodes for fimbrial adhesions have been detected in *S.* Enteritidis and cryptic bacteriophage has been seen within this locus in case of *S*. Typhi and *S.* Paratyphi A. On the other hand, *S.* Typhimurium has entirely different gene content. Because of these findings, the leuX locus represents a hot spot for the insertion of various mobile genetic elements [41].

#### **5.12 SPI11 and SPI12**

The SPI11 and SPI12 were identified in *Salmonella choleraesuis.* Both these islands shows properties of PAI such as association with bacteriophage genomes and tRNA genes. The low G + C content of 41.32% was seen for SPI11. The proteins encoded by these SPIs contributes to virulence of *Salmonella* but exact role is still not clear and awaits further characterization [42].

#### **5.13 SPI13 and SPI14**

SPI13 and SPI14 were first identified in avian adapted S. Gallinarum which is causative agent of typhoid in fowls. SPI13 is close to the tRNA pheV gene and is

*Pathogenicity Island in* Salmonella *DOI: http://dx.doi.org/10.5772/intechopen.96443*

composed of 18 ORFs while SPI14 is not associated with a tRNA gene and constitutes 6 ORFs. Both these islands are not present in *S*. Typhi and *S*. Paratyphi A but are seen in *S*. Typhimurium and *S*. Enteritidis. This may indicate a possible role of the loci in host specificity. The role of proteins encoded by these SPIs is not clear yet and requires further molecular characterization [43].

### **5.14 SPI15, SPI16, and SPI17**

SPI15, SPI16 and SPI17 were identified in S. Typhi using bioinformatics approach. All these exhibit association with tRNA genes. SPI16 and SPI17 encodes for genes that are responsible for LPS modification. There is presence of SPI15 in only S. Typhi isolate CT18 and role of its effecter proteins is not clear till date. SPI16 and SPI17 are seen in S. Typhi and most other S. enterica genome sequences [44].

**Table 4** summarizes the various SPIs.



#### **Table 4.**

*Summary of various* Salmonella *Pathogenicity Islands.*

#### **5.15 SGI1**

Strains showing resistance to multiple antibiotics is a usual phenomenon seen in pathogenic bacteria and is also mostly observed in S. enterica. Resistant *Salmonella* isolates harbor resistance plasmids of variable size and composition of resistance genes. A multidrug resistance phenotype conferred by '*Salmonella* genomic island 1' or SGI1 was recognized in epidemic strain S. Typhimurium DT104 by molecular testing though It can also be present in other strains as well. The SGI1 confers resistance to the antibiotics such as ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline [23].

#### **6. Conclusion**

PAI phenomenon frequently identified in pathogenic bacteria and encodes virulence genes which help pathogens to establish infections. The molecular characterization of individual virulence genes and genome sequences demonstrated large numbers of PAI in S. *enterica* serovars. Among various *Salmonella* pathogenicity islands, only SPI1 and SPI2 have well proven role in virulence while knowledge of the molecular function of the rest of the SPIs is lacking. Furthermore, molecular analysis of SPI is vital for improvement of prevention and treatment of *Salmonella* infection in human and animals. Also the varied degrees of disease severity and of bacterial pathogenesis can be explained better by understanding SPI.

*Pathogenicity Island in* Salmonella *DOI: http://dx.doi.org/10.5772/intechopen.96443*
